EPF-Lausanne/30 July 2009

From 2009.igem.org

(Difference between revisions)
(→Wet Lab)
 
(2 intermediate revisions not shown)
Line 27: Line 27:
==Wet Lab==
==Wet Lab==
-
==People in the lab==
+
Miniprep was done in the morning to extract the DNA for the LacI, death cassette and LovTap-Term parts.
 +
We decided to start over again for the LacI-RBS part but with different strategies and controls:
 +
 +
- strategy 1: insert the digested LacI into the RBS plasmid
 +
- strategy 2: insert the digested LacI and RBS into the death cassette plasmid (problem here: difficult to purify the RBS since the part is very small)
 +
 +
We did a PCR of LacI, GFP (for positive control) and a negative control. Then we purified the PCR products.
 +
 +
Digestion of:
 +
 +
- LacI PCR product with E/X
 +
 +
- RBS vector with E/X
 +
 +
- neg. control with E
 +
 +
- LacI PCR product with E/S
 +
 +
- LacI vector with E
 +
 +
- LacI vector with S
 +
 +
- death cassette vector with E/P
 +
 +
- RBS vector with X/P
 +
 +
- RBS vector with X
 +
 +
- RBS vector with P (--> there wasn't enough DNA left for this one)
 +
 +
(all these controls were meant to help check whether the digestions work well)
 +
 +
Treated RBS E/X vector and death cassette E/P vector with phosphatase, the RBS with P/X was put at 80°C for 20 min to inactivate enzymes. We then purified everything except the RBS P/X.
 +
 +
We ran all products on an agarose gel to see how the digestion worked.
 +
 +
We then went on with the ligation:
 +
 +
- RBS E/X (not gel-extracted) + LacI E/S
 +
- RBS E/X (gel-extracted) + LacI E/S
 +
- LacI E/S + RBS P/X + death cass. E/P
 +
- 2 neg. controls (vector only and no DNA)
 +
 +
We left the ligations overnight at 4°C.
 +
 +
==People in the lab==
 +
Basile, Gab, Christian, Mélanie, Nath
-
<html><center><a href="https://2009.igem.org/EPF-Lausanne/28_July_2009"><img src="https://static.igem.org/mediawiki/2009/thumb/6/61/Flèche_gauche.png/70px-Flèche_gauche.png"></a>   
+
<html><center><a href="https://2009.igem.org/EPF-Lausanne/29_July_2009"><img src="https://static.igem.org/mediawiki/2009/thumb/6/61/Flèche_gauche.png/70px-Flèche_gauche.png"></a>   
-
<a href="https://2009.igem.org/EPF-Lausanne/30_July_2009"><img src="https://static.igem.org/mediawiki/2009/thumb/5/5e/Fleche_droite.png/70px-Fleche_droite.png"></a></center></html>  
+
<a href="https://2009.igem.org/EPF-Lausanne/31_July_2009"><img src="https://static.igem.org/mediawiki/2009/thumb/5/5e/Fleche_droite.png/70px-Fleche_droite.png"></a></center></html>  
</div><div CLASS="epfl09bouchon"></div>
</div><div CLASS="epfl09bouchon"></div>

Latest revision as of 07:36, 3 August 2009



Contents

30 July 2009




Wet Lab

Miniprep was done in the morning to extract the DNA for the LacI, death cassette and LovTap-Term parts.

We decided to start over again for the LacI-RBS part but with different strategies and controls:

- strategy 1: insert the digested LacI into the RBS plasmid - strategy 2: insert the digested LacI and RBS into the death cassette plasmid (problem here: difficult to purify the RBS since the part is very small)

We did a PCR of LacI, GFP (for positive control) and a negative control. Then we purified the PCR products.

Digestion of:

- LacI PCR product with E/X

- RBS vector with E/X

- neg. control with E

- LacI PCR product with E/S

- LacI vector with E

- LacI vector with S

- death cassette vector with E/P

- RBS vector with X/P

- RBS vector with X

- RBS vector with P (--> there wasn't enough DNA left for this one)

(all these controls were meant to help check whether the digestions work well)

Treated RBS E/X vector and death cassette E/P vector with phosphatase, the RBS with P/X was put at 80°C for 20 min to inactivate enzymes. We then purified everything except the RBS P/X.

We ran all products on an agarose gel to see how the digestion worked.

We then went on with the ligation:

- RBS E/X (not gel-extracted) + LacI E/S - RBS E/X (gel-extracted) + LacI E/S - LacI E/S + RBS P/X + death cass. E/P - 2 neg. controls (vector only and no DNA)

We left the ligations overnight at 4°C.

People in the lab

Basile, Gab, Christian, Mélanie, Nath



Retrieved from "http://2009.igem.org/EPF-Lausanne/30_July_2009"